SLOB, a SLOWPOKE Channel Binding Protein, Regulates Insulin Pathway Signaling and Metabolism in Drosophila

There is ample evidence that ion channel modulation by accessory proteins within a macromolecular complex can regulate channel activity and thereby impact neuronal excitability. However, the downstream consequences of ion channel modulation remain largely undetermined. The Drosophila melanogaster large conductance calcium-activated potassium channel SLOWPOKE (SLO) undergoes modulation via its binding partner SLO-binding protein (SLOB). Regulation of SLO by SLOB influences the voltage dependence of SLO activation and modulates synaptic transmission. SLO and SLOB are expressed especially prominently in median neurosecretory cells (mNSCs) in the pars intercerebralis (PI) region of the brain; these cells also express and secrete Drosophila insulin like peptides (dILPs). Previously, we found that flies lacking SLOB exhibit increased resistance to starvation, and we reasoned that SLOB may regulate aspects of insulin signaling and metabolism. Here we investigate the role of SLOB in metabolism and find that slob null flies exhibit changes in energy storage and insulin pathway signaling. In addition, slob null flies have decreased levels of dilp3 and increased levels of takeout, a gene known to be involved in feeding and metabolism. Targeted expression of SLOB to mNSCs rescues these alterations in gene expression, as well as the metabolic phenotypes. Analysis of fly lines mutant for both slob and slo indicate that the effect of SLOB on metabolism and gene expression is via SLO. We propose that modulation of SLO by SLOB regulates neurotransmission in mNSCs, influencing downstream insulin pathway signaling and metabolism

Mechanisms of Two Modulatory Actions of the Channel-binding Protein Slob on the Drosophila Slowpoke Calcium-dependent Potassium Channel

Slob57 is an ion channel auxiliary protein that binds to and modulates the Drosophila Slowpoke calcium-dependent potassium channel (dSlo). We reported recently that residues 1–39 of Slob57 comprise the key domain that both causes dSlo inactivation and shifts its voltage dependence of activation to more depolarized voltages. In the present study we show that removal of residues 2–6 from Slob57 abolishes the inactivation, but the ability of Slob57 to rightward shift the voltage dependence of activation of dSlo remains. A synthetic peptide corresponding in sequence to residues 1–6 of Slob57 blocks dSlo in a voltage- and dose-dependent manner. Two Phe residues and at least one Lys residue in this peptide are required for the blocking action. These data indicate that the amino terminus of Slob57 directly blocks dSlo, thereby leading to channel inactivation. Further truncation to residue Arg16 eliminates the modulation of voltage dependence of activation. Thus these two modulatory actions of Slob57 are independent. Mutation within the calcium bowl of dSlo greatly reduces its calcium sensitivity (Bian, S., I. Favre, and E. Moczydlowski. 2001. Proc. Natl. Acad. Sci. USA. 98:4776–4781). We found that Slob57 still causes inactivation of this mutant channel, but does not shift its voltage dependence of activation. This result confirms further the independence of the inactivation and the voltage shift produced by Slob57. It also suggests that the voltage shift requires high affinity Ca2+ binding to an intact calcium bowl. Furthermore, Slob57 inhibits the shift in the voltage dependence of activation of dSlo evoked by Ca2+, and this inhibition by Slob57 is greater at higher free Ca2+ concentrations. These results implicate distinct calcium-dependent and -independent mechanisms in the modulation of dSlo by Slob

Pattern of distribution and cycling of SLOB, Slowpoke channel binding protein, in Drosophila

BACKGROUND: SLOB binds to and modulates the activity of the Drosophila Slowpoke (dSlo) calcium activated potassium channel. Recent microarray analyses demonstrated circadian cycling of slob mRNA. RESULTS: We report the mRNA and protein expression pattern of slob in Drosophila heads. slob transcript is present in the photoreceptors, optic lobe, pars intercerebralis (PI) neurons and surrounding brain cortex. SLOB protein exhibits a similar distribution pattern, and we show that it cycles in Drosophila heads, in photoreceptor cells and in neurosecretory cells of the PI. The cycling of SLOB is altered in various clock gene mutants, and SLOB is expressed in ectopic locations in tim(01 )flies. We also demonstrate that SLOB no longer cycles in the PI neurons of Clk(jrk )flies, and that SLOB expression is reduced in the PI neurons of flies that lack pigment dispersing factor (PDF), a neuropeptide secreted by clock cells. CONCLUSIONS: These data are consistent with the idea that SLOB may participate in one or more circadian pathways in Drosophila

Defects in synapse structure and function precede motor neuron degeneration in Drosophila models of FUS-related ALS.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease that leads invariably to fatal paralysis associated with motor neuron degeneration and muscular atrophy. One gene associated with ALS encodes the DNA/RNA-binding protein Fused in Sarcoma (FUS). There now exist two Drosophila models of ALS. In one, human FUS with ALS-causing mutations is expressed in fly motor neurons; in the other, the gene cabeza (caz), the fly homolog of FUS, is ablated. These FUS-ALS flies exhibit larval locomotor defects indicative of neuromuscular dysfunction and early death. The locus and site of initiation of this neuromuscular dysfunction remain unclear. We show here that in FUS-ALS flies, motor neuron cell bodies fire action potentials that propagate along the axon and voltage-dependent inward and outward currents in the cell bodies are indistinguishable in wild-type and FUS-ALS motor neurons. In marked contrast, the amplitude of synaptic currents evoked in the postsynaptic muscle cell is decreased by \u3e80% in FUS-ALS larvae. Furthermore, the frequency but not unitary amplitude of spontaneous miniature synaptic currents is decreased dramatically in FUS-ALS flies, consistent with a change in quantal content but not quantal size. Although standard confocal microscopic analysis of the larval neuromuscular junction reveals no gross abnormalities, superresolution stimulated emission depletion (STED) microscopy demonstrates that the presynaptic active zone protein bruchpilot is aberrantly organized in FUS-ALS larvae. The results are consistent with the idea that defects in presynaptic terminal structure and function precede, and may contribute to, the later motor neuron degeneration that is characteristic of ALS

Slob, a Novel Protein that Interacts with the Slowpoke Calcium-Dependent Potassium Channel

AbstractSlob, a novel protein that binds to the carboxy-terminal domain of the Drosophila Slowpoke (dSlo) calcium-dependent potassium channel, was identified with a yeast two-hybrid screen. Slob and dSlo coimmunoprecipitate from Drosophila heads and heterologous host cells, suggesting that they interact in vivo. Slob also coimmunoprecipitates with the Drosophila EAG potassium channel but not with Drosophila Shaker, mouse Slowpoke, or rat KV1.3. Confocal fluorescence microscopy demonstrates that Slob and dSlo redistribute in cotransfected cells and are colocalized in large intracellular structures. Direct application of Slob to the cytoplasmic face of detached membrane patches containing dSlo channels leads to an increase in channel activity. Slob may represent a new class of multi-functional channel-binding proteins

Regulation of intracellular free arachidonic acid in Aplysia nervous system

We have studied the regulation of arachidonic acid (AA) uptake, metabolism, and release in Aplysia nervous system. Following uptake of [ 3 H]AA, the distribution of radioactivity in intracellular and extracellular lipid pools was measured as a function of time in the presence or absence of exogenous AA. The greatest amount of AA was esterified into phosphatidylinositol (relative to pool size). We found that the intracellular free AA pool underwent rapid turnover, and that radioactive free AA and eicosanoids were released at a rapid rate into the extracellular medium, both in the presence and absence of exogenous AA. Most of the released radioactivity originated from phosphatidylinositol.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/48020/1/232_2005_Article_BF01868464.pd